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標題: | 高毒性克雷伯氏肺炎桿菌菌株演化分析:多位點序列分型(MLST)、K基因型、O基因型、與其他毒性因子的整合分析 Evolution of Hypervirulent Klebsiella pneumoniae: an Integrative Analysis of Multilocus sequence typing (MLST), K genotypes, O genotypes, and other Virulent factors |
作者: | 王思惠 Szu-Hui Wang |
指導教授: | 方啟泰 |
關鍵字: | 高毒性克雷伯氏肺炎桿菌,多位點序列分型,細菌性肝膿瘍,致病機轉,病例對照研究, hypervirulent Klebsiella pneumoniae,multilocus sequence typing,pyogenic liver abscess,pathogenesis,case-control study, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 背景
侵襲性克雷伯氏肺炎桿菌感染(以肝膿瘍最多,可併發眼內炎、腦膜炎、壞死性筋膜炎等重症)是臺灣最常見的急性感染重症之一。雖然目前已發現許多重要毒性基因型(莢膜基因型K1, K2, K5, K20, K54, 及酯多醣基因型O1)及毒性基因rmpA與補鐵系統 (kfu, aerobactin, yersiniabactin, and salmochelin gene clusters),迄今「高毒性克雷伯氏肺炎桿菌」 (hypervirulent Klebsiella pneumoniae) 仍尚未有診斷標準。毒性株除了須考慮K, O基因型及其他毒性基因之組合,尚須考慮菌株同源性 (clonality)。 研究目的 本研究藉由分析臨床克雷伯氏肺炎桿菌菌株演化起源與毒性基因或基因型(K, O基因型、rmpA與補鐵系統)之關聯性,建立高毒性克雷伯氏肺炎桿菌的診斷標準。 方法 本研究自常規收集臨床檢體之克雷伯氏肺炎桿菌菌株庫,選取177株分離自肝膿瘍病人之菌株,以及一病例對照研究之269株,包括90株分離自社區感染克雷伯氏肺炎桿菌肝膿瘍之病例組病人以及179株分離自同樣為社區感染克雷伯氏肺炎桿菌但未發生肝膿瘍之對照組病人的菌株。藉由法國巴斯德研究所開發的長程演化分析工具Multilocus Sequence Typing (MLST) 進行克雷伯氏肺炎桿菌菌株之演化分析,並使用eBURST軟體分析MLST之演化起源,釐清MLST之菌株演化溯源 (clonal lineage)以及其他毒性基因/基因型之間的關聯性,定義菌株之染色體clone。針對細菌質體的部分則以比較基因體方式,分析不同染色體clone菌株之質體上的毒性基因攜帶模式。其後將演化分析所定義之菌株染色體clone以及質體毒性基因攜帶模式藉由病例對照研究中的危險因子分析,發展高毒性克雷伯氏肺炎桿菌菌株的診斷標準。並藉由多種評估交互作用之指標,如synergy index、RERI以及互補對數迴歸等方法,檢驗免疫力較弱之糖尿病患者與菌株毒性之間是否存在生物性交互作用或因果機轉交互作用。 結果 本研究進行446株克雷伯氏肺炎桿菌之演化分析,並發現有三個主要的clonal complex (CC),分別是由ST23、ST322、ST3126、ST3127、ST322以及ST218組成,主要為K1基因型之CC23,以及由ST65、ST25、ST375、ST814以及ST2271組成之CC65和由ST86、ST373、ST2272組成之CC86。而CC65與CC86皆為K2基因型菌株,且這三者之O基因型約95%-100%為O1基因型,其他毒性基因於三個CC之間的分布亦有顯著差異,故定義菌株染色體clone包括:K1-O1-CC23、K2-O1-CC65以及K2-O1-CC86。另外,由四個在質體上之毒性基因 (iucB、p-iroN、p-rmpA、rmpA2)的分布情況將其攜帶毒性基因的模式分類為完整四個基因的pVLP-full、攜帶部分基因之pVLP-partial以及完全未帶有四個毒性基因之pVLP-null。由比較基因體之分析結果發現即便是有相同的染色體基因型,仍可能有不同的毒性基因攜帶模式。故將染色體與質體的基因分開視為獨立的兩個因子納入之後的統計分析。 在病例對照研究中,多變項羅吉斯迴歸顯示K1-O1-CC23 (adj. OR=6.81, p<0.0001)、K2-O1-CC65 (adj. OR=4.65, p=0.0099)以及pVLP-full (adj. OR=3.948, p=0.0012)是造成克雷伯氏肺炎桿菌肝膿瘍的獨立危險因子,但其他宿主潛在疾病皆非獨立之危險因子,而肝硬化在迴歸模型中為保護因子(adj. OR=0.202, p=0.0169)。評估生物性交互作用之指標,不論synergy index、RERI或是互補對數迴歸皆無法直接證實個別的染色體clone與糖尿病之間存在顯著的生物性交互作用或因果機轉交互作用。 結論 本研究歸納出三種造成肝膿瘍之高毒性克雷伯氏肺炎桿菌菌株:包括兩個染色體clone K1-O1-CC23、K2-O1-CC65以及帶有四個毒性基因之pVLP-full質體,於克雷伯氏肺炎桿菌肝膿瘍之致病機轉扮演非常重要的角色。此外,本研究結果也解釋了K2菌株在MLST上的多元性,K2-O1-CC86菌株之染色體clone毒性較弱,其造成侵襲性感染之毒性來自於pVLP,同時也解釋了臨床上某些病人感染非K1、K2基因型的菌株卻仍發生肝膿瘍的情況。從各項交互作用評估方法無法直接證實個別的染色體clone以及pVLP與糖尿病的交互作用,然而本研究族群之糖尿病盛行率明顯較一般族群高,故我們以純病例方法 (case-only method) 探討糖尿病是否於克雷伯氏肺炎桿菌致病機轉的其他階段造成影響,並證實糖尿病為造成一般民眾發生克雷伯氏肺炎桿菌感染的危險因子。 Background Tissue-invasive Klebsiella pneumoniae infections (pyogenic liver abscess, endophthalmitis, meningitis, necrotizing fasciitis, etc.) is one of the leading infectious diseases in Taiwan. Prior studies identified several important virulence genes and genotypes, including capsular polysaccharide genotypes K1, K2, K5, K20, K54, lipopolysaccharides genotype O1, rmpA and iron-uptake systems (kfu, aerobactin, yersiniabactin, and salmochelin) gene clusters. Nevertheless, there is still no diagnostic criteria for hypervirulent K. pneumoniae strains, for which the definition of hypervirulent clones needs to be based on clonality in bacterial evolution. Aims This study aims to establish the bacteriological criteria for hypervirulent K. pneumoniae, by analyzing the association between the phylogenetic origins and virulence genes/genotypes (K, O, rmpA, iron-uptake systems) in clinical K. pneumoniae strains. Materials and Methods We have selected 177 K. pneumonia strains isolated from pyogenic liver abscess (PLA) patients and 269 strains from a case-control study, including 90 strains isolated from PLA cases and 179 strains from controls who were patients with community-acquired K. pneumonia isolates but did not develop PLA. We used multilocus sequence typing (MLST), developed by Pasteur Institute (Paris, France), to analyze the phylogenetic origins of K. pneumonia strains, and trace the phylogenetic origins of MLST types by eBURST. Then we could establish the K. pneumonia chromosome clones by clarifying the correlation between MLST clonal lineage and other virulence genes/ genotypes. As for K. pneumonia plasmids, we analyzed the patterns of virulence genes on plasmids by comparative genomic analysis. Afterwards, we would use evolution-based definition for K. pneumoniae clones to develop diagnostic criteria for hypervirulent K. pneumoniae clones based on risk factors in case-control study. We have also examined the biological interactions between DM and K. pneumoniae clones by synergy index and RERI, and test the causal mechanistic interaction by complementary log regression. Results A total of 446 K. pneumoniae strains were included in the phylogenic analysis. We found three major clonal complexes including (1) CC23 composed of ST23, ST322, ST3126, ST322 and ST218, most of them belong to K1 genotype; (2) CC65 composed of ST65, ST25, ST375, ST814 and ST2271; (3) CC86 composed of ST86, ST373 and ST2272, all of the strains in CC65 and CC86 are K2 genotype. These three CCs have a high proportion (95%-100%) of O1 genotype and with a significant different distribution of other virulent genes. Therefore, we defined the chromosome clones are K1-O1-CC23, K2-O1-CC65, and K2-O1-CC86. In addition, by the distribution of 4 virulence genes (iucB, p-iroN, p-rmpA, rmpA2), we classified plasmid into pVLP-full (carried all of virulence genes), pVLP-partial (carried part of virulent genes) and pVLP-null (carried none of virulent genes). Through comparative genomic analysis, we found that even with the same chromosome clones, K. pneumoniae might still have different patterns of virulence genes on the plasmids, and we should take chromosome clones and plasmid as separated variables in the statistical analysis. In case-control study, multivariable logistic regression showed that K1-O1-CC23 (adj. OR=6.81, p<0.0001), K2-O1-CC65 (adj. OR=4.65, p=0.0099) and pVLP-full (adj. OR=3.948, p=0.0012) were independent risk factors for K. pneumoniae PLA. Other host underlying disease were not risk factors and liver cirrhosis is a protective factor in the result. We examined the effect of interaction between DM and K. pneumoniae clones by synergy index, RERI and complementary log regression, but none of them showed a significant result. Conclusions The present study demonstrated that there are three significant hypervirulent K. pneumoniae clones play a predominant role in the pathogenesis of PLA, including two chromosome clones K1-O1-CC23, K2-O1-CC65, and plasmid with full of four virulence genes (pVLP-full). The results also explained the diversity of STs of K2 genotypes. K2-O1-CC86 is a relatively avirulent chromosome clone, its virulence for causing invasive infection might come from pVLP, it may simultaneously explained the situation that some patients infected by non-K1 or non-K2 genotype strains but still developed PLA. The prevalence of DM in our cases and controls are much higher than general population, we used the case-only method to analyze the effect of DM in other stages of the pathogenesis of K. pneumoniae PLA and found that DM will increase the risk of developing K. pneumoniae infections for people who were not exposed or already colonized by K. pneumoniae but not developing infections yet. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77457 |
DOI: | 10.6342/NTU201802887 |
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顯示於系所單位: | 流行病學與預防醫學研究所 |
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